DOI 10.1007/s10600-015-1290-z Chemistry of Natural Compounds, Vol 51, No 2, March, 2015 PHYTOCHEMICAL CONSTITUENTS OF Artemisia dubia var longeracemosa FORMA tonkinensis Phan Minh Giang,1* Tran Thi Thanh Nhan,1 Truong Thi To Chinh,1 Phan Tong Son,1 and Katsuyoshi Matsunami2 Higher plants are able to produce a vast number of secondary metabolites that have been demonstrated to display a variety of biological actions The therapeutic efficacy of medicinal plants is believed to be controlled by complex mixtures of different compounds, which may be active, synergistic, complementary, antagonistic, or toxic substances Therefore, chemical knowledge of medicinal plants is a focal point in the modernization of traditional medicines in many developing countries Artemisia is a large, widespread, and medicinally important genus of the family Asteraceae Intensive chemical and biological investigations have recognized Artemisia plants as generous sources of unique biologically active natural compounds such as sesquiterpene lactones and polyacetylenic compounds [1] A dubia Wall ex Besser is distributed in Bhutan, China, India, Japan, Nepal, and Thailand and is recorded as a medicinal plant of these countries in the treatment of gastric problems, intestinal worms, and skin infections [2, 3] Very few phytochemical studies have been performed on this species In a study of A dubia of China, oxygenated guaianolides, which showed no or only weak cytotoxic activity against several human cancer cell lines, were isolated [4] From A dubia of Korea sesquiterpene lactones have not been isolated yet; however, dicaffeoylquinic acids with potent inhibitory activity on recombinant human AKR1B10 were found [5] A dubia Wall ex Bess var longeracemosa Pamp forma tonkinensis Pamp (Vietnamese name: Thanh cao Bac Bo) of Vietnam has not been investigated chemically and is the subject of our present investigation The dried leaves of A dubia var longeracemosa were extracted with MeOH, and the resulting MeOH extract was sequentially fractionated using solvents of increasing polarities to give n-hexane-, CH2 Cl2-, and EtOAc-soluble fractions The n-hexane- and CH2Cl2-soluble fractions were fractionated by silica gel CC to give pool fractions of different polarities Repeated silica gel CC and FC of the fractions obtained afforded compounds 1–12 They are two oleanene-type triterpenoids: calotropoleanyl ester (1) [6] and its deacetylated derivative, 13(18)-oleanen-3E-ol (2) [6]; an ursane-type triterpenoid: D -amyrin (3) [7]; four fatty acids: nonacosanoic acid (4), docosanoic acid (5), tetracosanoic acid (6), and palmitic acid (10) [8]; two phytosterols: E-sitosterol (7) and E-sitosterol 3-O-E-D-glucopyranoside (12) [7]; two acyl glycerols: 1-O-(palmitoyl)glycerol (8) and 1-O-(stearoyl)glycerol (9) [9, 10]; and an indole derivative, 6-methoxy-1H-indole-3-methylcarboxylate (11) [11] The known compounds 1–10 and 12 were determined by comparing their spectroscopic data with the reported literature values [6–10] Compounds 1, 2, 8, and were isolated for the first time from Artemisia genus Compound 11 is only known synthetically [11]; 11 was isolated for the first time from nature in this study Compound 11 was isolated as a white amorphous powder, and its molecular formula was suggested to be C11H11O3N on the basis of ESI-TOF-MS In the IR spectrum of 11 a secondary amine group (Qmax 3239 cm–1), aromatic rings (Qmax 1632, 1584, 1532, and 1449 cm–1), and an aromatic ester (Qmax 1660 and 1285 cm–1) were present The 1H, 13C NMR, and HSQC spectroscopic data of 11 revealed the presence of a substituted indole ring (GC 95.0, 108.0, 111.8, 119.9, 122.1, 130.1, 136.9, and 157.1) [12], an aromatic methoxy group [GH 3.82 (3H, s); GC 55.6 (q)], and a methylcarboxylate group [GH 3.91 (3H, s); GC 165.5 (s) and 51.1 (q)] In order to determine the substitution pattern of the indole ring, the NOESY spectrum of 11 was recorded (Fig 1) 1) Faculty of Chemistry, VNU University of Sciences, Vietnam National University, Hanoi, 19 Le Thanh Tong Street, Hanoi, Vietnam, e-mail: phanminhgiang@yahoo.com; 2) Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan Published in Khimiya Prirodnykh Soedinenii, No 2, March–April, 2015, pp 329–330 Original article submitted May 19, 2013 378 0009-3130/15/5102-0378 2015 Springer Science+Business Media New York O OCH3 N H3CO H 11 Fig Structure HMBC correlations of 11 NOEs were observed between each pair of protons in the following sequence: from H-2 [GH 7.78 (1H, d, J = 2.5 Hz)] to a NH-group [GH 8.73 (1H, br.s)]; from the NH-group to H-7 [GH 6.85 (1H, d, J = 2.5 Hz)]; from H-7 to CH3O-6 [GH 3.82 (3H, s)]; from CH3O-6 to H-5 [G H 6.92 (1H, dd, J = 2.5, 8.5 Hz)]; and from H-5 to H-4 [GH 8.04 (1H, d, J = 8.5 Hz, H-4)] Thus the location of the aromatic methoxy group was established at C-6, which is in good agreement with the ortho coupling of H-4 and H-5 (J = 8.5 Hz) and meta coupling of H-5 and H-7 (J = 2.5 Hz) Finally, HMBC long-range correlations (Fig 1) determined the structure of 11 to be 6-methoxy-1H-indole-3-methylcarboxylate since H-2 showed correlation to the carbonyl carbon at GC 165.5 and methoxy protons at GH 3.91 displayed correlation to the same carbon General Experimental Procedures Optical rotations were measured on a JASCO P-1030 digital polarimeter FT-IR spectra were recorded on a Horiba FT-710 spectrophotometer ESI-MS spectra were recorded on a Thermo Scientific LTQ Orbitrap XL mass spectrometer ESI-TOF-MS spectra were recorded on an Applied Biosystem QSTAR XL mass spectrometer H and 13C NMR spectra were recorded on a Bruker Avance 500 NMR spectrometer Merck silica gel (0.063–0.200 mm, 0.063–0.100 mm, 0.040–0.063 mm, and 0.015–0.040 mm) was used for open-column chromatography (CC) and flash chromatography (FC) Thin-layer chromatography (TLC) was carried out on Merck TLC silica gel 60 F254 and detected by spraying with 1% vanillin in concentrated H2SO4, followed by heating on a hot plate Plant Material The leaves of A dubia Wall ex Bess var longeracemosa Pamp forma tonkinensis Pamp (Asteraceae) were collected in Kontum Province, Vietnam in August 2008 The plant was identified by Mr Nguyen Quoc Binh, a botanist of the Institute of Biological Resources and Ecology, Vietnam Academy of Science and Technology, Hanoi, Vietnam A voucher specimen of the plant (voucher number QB 623) has been deposited at the same Institute Extraction and Isolation The powdered dried leaves of A dubia var longeracemosa (5 kg) were extracted with MeOH at room temperature (three times, each time for three days) and the resultant MeOH extract was evaporated under reduced pressure The MeOH extract was fractionated by successive partition between H2O and n-hexane, CH2Cl2 , and EtOAc to give the corresponding soluble fractions Part of the n-hexane-soluble fraction (45 g) was subjected to silica gel CC using n-hexane–acetone (19:1, 9:1, 6:1, 3:1, 2:1, and 1:1) to give eleven fractions Fractions and gave (1.6 g) and a mixture of and (4.6 g), respectively, on washing with MeOH Faction was washed with n-hexane to give (759 mg) Silica gel CC eluting with n-hexane–acetone (15:1, 12:1, 9:1, and 6:1) gave (50 mg) from fraction 5, and (460 mg) and (2.1 g) from fraction Repeated chromatography on silica gel CC eluting with n-hexane–EtOAc (19:1, 9:1, 6:1, 3:1, and 1:1), reversed phase (RP)-18 with MeOH–H2O (7:3 and 4:1), and silica gel CC with n-hexane–acetone (3:1) gave a mixture of and (40 mg) Part of the CH2Cl2-soluble fraction (15 g) was subjected to silica gel CC using n-hexane–acetone (90:1, 49:1, 19:1, 9:1, 6:1, 3:1, and 1:1) to give 10 fractions Fractions and were combined and subjected to silica gel FC using n-hexane–EtOAc (29:1, 19:1, and 9:1) to give (10 mg) Fraction was separated by silica gel FC with n-hexane–EtOAc (29:1, 19:1, and 9:1) to give (10.4 mg) and (17 mg) Fraction was separated by silica gel FC eluting successively with n-hexane–EtOAc (19:1 and 9:1), CH2Cl2–EtOAc (70:1, 49:1, 29:1, 19:1, 9:1, and 4:1), and CH2Cl2–EtOAc (90:1 and 70:1) to give 10 (11.8 mg) Fraction was separated three times by silica gel FC eluting with n-hexane–EtOAc (7:1, 5:1, 4:1, 3:1, 2:1, and 1:1), n-hexane–EtOAc (5:1, 4:1, 3:1, and 2:1), and CH2Cl2–EtOAc (90:1, 70:1, and 15:1) to give 11 (44 mg) Fraction 10 was purified by silica gel FC with CH2Cl2–MeOH (9:1 and 7:1) to give 12 (29.7 mg) 6-Methoxy-1H-indole-3-methylcarboxylate (11) White amorphous powder IR (film, Qmax, cm–1): 3230, 1660, 1632, 1584, 1532, 1449, 1285, 1196, 1156 1H NMR (500 MHz, CDCl3, G, ppm, J/Hz): 3.82 (3H, s, CH3O-6), 3.91 (3H, s, CH3CO2-3), 6.85 (1H, d, J = 2.5, H-7), 6.92 (1H, dd, J = 2.5, 8.5, H-5), 7.78 (1H, d, J = 2.5, H-2), 8.04 (1H, d, J = 8.5, H-4), 8.73 (1H, br.s, NH) 13C NMR (125 MHz, CDCl3, G, ppm): 51.1 (CH3CO2-3), 55.6 (CH3O-6), 95.0 (C-7), 108.0 (C-3), 111.8 (C-5), 119.9 (C-3a), 122.1 (C-4), 130.1 (C-2), 136.9 (C-7a), 157.1 (C-6), 165.5 (CH3CO2-3) Positive-ion ESI-TOF-MS m/z 228.1236, C11H11NO3Na, [M + Na]+ 379 ACKNOWLEDGMENT This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.01-2012.10 REFERENCES 10 11 12 380 R X Tan, W F Zheng, and H Q Tang, Planta Med., 64, 295 (1998) M Ashraf, M Q Hayat, S Jabeen, N Shaheen, M A Khan, and G Yasmin, J Med Plants Res., 4, 112 (2010) M Q Hayat, M A Khan, M Ashraf, and S Jabeen, Ethnobot Res Appl., 7, 147 (2009) Z S Huang, Y H Pei, C M Liu, S Lin, J Tang, D S Huang, T F Song, L H Lu, Y P Gao, and W D Zhang, Planta Med., 76, 1710 (2010) H J Lee, J Y Lee, S M Kim, C W Nho, S H Jung, D G Song, C Y Kim, and C H Pan, J Korean Soc Appl Biol Chem., 53, 826 (2010) S H Ansari and M Ali, Indian J Chem., 39B, 287 (2000) L J Goad and T Akihisha, Analysis of Sterols, Chapman & Hall, London, 1997 G C Levy, Topics in Carbon-13 NMR Spectroscopy, Vol 2, John Wiley & Sons, New York, 1976 S H Qi, S Zhang, J S Huang, Z H Xiao, J Wu, and L J Long, Chem Pharm Bull., 52, 986 (2004) C C Yu, Y S Lee, B S Cheon, and S H Lee, Bull Korean Chem Soc., 24, 1229 (2003) T Masahiro and F Keiichiro, Heterocycles, 31, 1431 (1990) E Pretsch, P Buhlmann, and M Badertscher, Structure Determination of Organic Compounds, Springer, Berlin, 2009 ... vanillin in concentrated H2SO4, followed by heating on a hot plate Plant Material The leaves of A dubia Wall ex Bess var longeracemosa Pamp forma tonkinensis Pamp (Asteraceae) were collected in... long-range correlations (Fig 1) determined the structure of 11 to be 6-methoxy-1H-indole-3-methylcarboxylate since H-2 showed correlation to the carbonyl carbon at GC 165.5 and methoxy protons at GH 3.91... H-4)] Thus the location of the aromatic methoxy group was established at C-6, which is in good agreement with the ortho coupling of H-4 and H-5 (J = 8.5 Hz) and meta coupling of H-5 and H-7 (J